Method for the preparation of (3S, 3S′) 4,4′-disulfanediylbis (3-aminobutane 1-sulfonic acid)

ABSTRACT

The present invention relates to a new method for the preparation of (3S,3S′) 4,4′-disulfanediylbis(3-aminobutane 1-sulfonic acid) in five steps from (S)-ethyl 2-(benzyloxycarbonylamino)-4-(neopentyloxysulfonyl)butanoate A.

This application is the U.S. national phase of International ApplicationNo. PCT/EP2011/067524, filed 7 Oct. 2011, which designated the U.S. andclaims priority to EP Application No. 10306099.2, filed 7 Oct. 2010, theentire contents of each of which are hereby incorporated by reference.

The present invention relates to a new method for the preparation of(3S,3S′) 4,4′-disulfanediylbis(3-aminobutane 1-sulfonic acid) in fivesteps from (S)-ethyl2-(benzyloxycarbonylamino)-4-(neopentyloxysulfonyl)butanoate A. (3 S,3S′) 4,4′-disulfanediylbis(3-aminobutane 1-sulfonic acid) is referred toas “Compound I” in the present invention.

Compound I is a dimer of the selective aminopeptidase A (APA) inhibitor3-amino 4-mercaptobutanesulfonic acid (also called EC33 in previousdocuments), generated by creating a disulfide bond between thiolextremities of two 3-amino 4-mercaptobutanesulfonic acid molecules.Dimerisation affords a molecule more amenable to cross the blood-brainbarrier as a prodrug. Compound I (also called RB150 in previousdocuments) has been proven to be an efficient anti-hypertensor agent, asdescribed by Bodineau et al. in Hypertension 2008 51, 1318-1325.

Compound I and use thereof as anti-hypertensor were disclosed in thepatent application WO2004/007441. The example of process provided inthis document to synthesise compound I allows its formation, in 6 steps,from L-Homoserin. Technical specifications, particularly numbers ofequivalents, solvents and/or purification techniques involved in thisprocess, do not allow it to be efficiently and easily converted into anindustrial scale.

A permanent aim in organic synthesis is to create synthesis processesthat can be transposed into industrial conditions. In order to meetrequirements for industrial processes, different parameters of thesynthesis are to be optimized. First, solvents must be as littlevolatile as possible, in order to be easily recoverable. Thus,chlorinated volatile solvents, e.g. dichloromethane, chloroform and/orcarbon tetrachloride, are preferably avoided. In addition, the numbersof equivalents of reagents required are preferably limited, thetemperatures involved preferably remain in an easily accessible range,and easy to proceed purification steps should be privileged. Finally,reaction mixtures and isolated product are preferably thermally stable.

Current Good Manufacturing Practice (c-GMP) has been defined forpreparation of drug products for administration to humans or animals.GMP regulations require a quality approach to manufacturing, enablingcompanies to minimize or eliminate instances of contamination, mixups,and errors. GMP regulations address issues including recordkeeping,personnel qualifications, sanitation, cleanliness, equipmentverification, process validation, and complaint handling.

To the Applicant knowledge, no industrially applicable process tosynthesise compound I has been described so far.

Hence, an object of the present invention is to provide a process forpreparing compound I that can be adapted easily and efficiently toindustrial scale, as compared to the process of the prior-art, whereintoxic solvents, such as dimethylformamide, and column chromatography areused.

Moreover, since a highly pure form, typically greater than 99.5 percent,of any drug is generally required for human treatment, a method thatcombines the control of the formation of isomers and a facile finalpurification is particularly advantageous.

METHOD

The present invention relates to a new method for the preparation ofcompound I, more particularly in 5 steps, from (S)-ethyl2-(benzyloxycarbonylamino) 4-(neopentyloxysulfonyl)butanoate A. Scheme 1illustrates the successive steps leading from A to compound I.

Unless otherwise stated, the following abbreviations and denominationsare used throughout the description and claims of the present invention:

Et=ethyl; tBu=tert-butyl; CH₂-tBu=neopentyl=2,2-dimethylpropyl

Cbz=Carbobenzyloxy

Ms=mesyl=SO₂CH₃

TFA=trifluoroacetic acid

THF=tetrahydrofuran

MTBE=methyltert-butylether

HPLC=High Performance Liquid Chromatography

ee=enantiomeric excess

Each reaction described herein may be performed in solid phase or inliquid phase. Liquid phase reactions may be preferably performed in asolvent selected from organic or aqueous solvents, for example THF,ethanol, chloroform, MTBE, toluene, acetone, TFA, and/or anisole.

The first object of the present invention relates to a general methodfor the preparation of compound I from A, comprising the followingsteps:

-   -   (a) reducing the ethyl ester of A, to give (S)-neopentyl        3-(benzyloxycarbonylamino) 4-hydroxybutane 1-sulfonate B;    -   (b) reacting the alcohol B with methanesulfonic anhydride or        methanesulfonyl chloride in presence of a base, to give        (S)-neopentyl 3-(benzyloxycarbonylamino)        4-(methylsulfonyloxy)butane 1-sulfonate C;    -   (c) reacting the mesylated alcohol C with potassium thioacetate,        to give (S) 2-(benzyloxycarbonylamino)        4-(neopentyloxysulfonyl)butyl thioacetate D;    -   (d) dimerizing D to give (3S,3S′) neopentyl        4,4′-disulfanediylbis(3-(benzyloxycarbonylamino)butane        1-sulfonate) E; and    -   (e) deprotecting sulfonic ester and amine groups of E, to give        (3S,3S′) 4,4′-disulfanediylbis(3-aminobutane 1-sulfonic acid)        compound I.

The above described method is referred to as “the general method” in thepresent description.

Preferably, step (a) may be performed by reacting A with a reducingagent-solvent couple selected from NaBH₄/LiCl—mixture of THF andethanol, preferably in 1:1 volume ratio, and LiBH₄—THF, more preferablyLiBH₄—THF. The reaction may be performed at a temperature from about 0°C. to about 25° C., preferably from about 20° C. to about 25° C.

More preferably, step (a) may be performed by reacting A with LiBH₄—THF,at a temperature from about 20° C. to about 25° C.

The use of LiBH₄, that is soluble and stable in THF, represents anundeniable safety improvement; particularly, it allows use of neat THFas solvent and hence avoids liberation of hydrogen gas due todecomposition of sodium borohydride in ethanol.

Preferably, step (b) may be performed in presence of triethylamine. Thereaction may be performed in a solvent selected from chloroform and amixture of MTBE and toluene, preferably a mixture of MTBE and toluene,preferably in 3:2 volume ratio. The reaction may be performed at atemperature from about −10° C. to about 10° C., preferably from about 5°C. to about 10° C.

Industrial transposition of a synthesis requires volatile solvents to bepreferably replaced with less volatile, and/or easier to recoversolvents. More preferred conditions for step (b) in this inventioninclude replacement of chloroform with a less volatile and/or easier torecover solvent, such as a mixture of MTBE and toluene in 3:2 volumeratio.

More preferably, step (b) may be performed in presence of triethylaminein a mixture of MTBE and toluene in 3:2 volume ratio, at a temperaturefrom about 5° C. to about 10° C.

Preferably, step (c) may be performed in a solvent selected from ethanoland acetone, preferably acetone. The reaction may be performed at atemperature from about 15° C. to about 25° C.

More preferably, step (c) may be performed in acetone, at a temperaturefrom about 15° C. to about 25° C.

Preferably, step (d) may be performed by first contacting D with sodiumhydroxide. The obtained mixture may then be reacted with iodine. Thesolvent may be ethanol. The reaction may be performed at a temperaturefrom about 15° C. to about 25° C.

Preferably, step (e) may be performed by stirring E in a mixture of TFAand anisole. More preferably, step (e) may be performed by stirring E ina refluxing mixture of TFA and anisole, preferably in 5:1 volume ratio.

A most preferred form of the present invention is the general methoddescribed above, wherein:

-   -   step (a) is performed by reacting A with LiBH₄—THF, at a        temperature from about 20° C. to about 25° C.;    -   step (b) is performed in presence of triethylamine, in a mixture        of MTBE and toluene in 3:2 volume ratio, at a temperature from        about 5° C. to about 10° C.;    -   step (c) is performed in acetone, at a temperature from about        15° C. to about 25° C.;    -   step (d) is performed by first contacting D with sodium        hydroxide, in ethanol, at a temperature from about 15° C. to        about 25° C. and then reacting the obtained mixture with iodine,        in ethanol, at a temperature from about 15° C. to about 25° C.;        and    -   step (e) is performed by stirring E in a refluxing mixture of        TFA and anisole in 5:1 volume ratio.

This method comprises (a) to (e) optimised steps for industrialapplication, in particular steps (b) to (e) are even compliant to c-GMPrequirements.

Industrial transposition of a synthesis requires parameters to beoptimised. In particular, high enthalpy reactions are preferablyavoided. High purity levels are preferred for products. Isolatedproducts are preferably thermally stable.

Table 1 provides the reaction enthalpies, the purities (determined byHPLC and expressed as molar percentages) and the stabilities of theproducts of each step for this preferred method.

TABLE 1 Purity Enthalpies Stability step   94.0%ΔrH_((addition onto LiBH) ₄ ₎ = Melting at 53° C. (a) −235 kJ/mol ee 98%ΔrH_((HCl hydrolysis)) = −145 kJ/mol step     98% ΔrH = −253 kJ/molMelting at 66° C. (b) Decomposition at 103° C. step     98% ΔrH = −145kJ/mol Melting at 72° C. (c) Decomposition at 154° C. step     97%ΔrH_((addition of NaOH)) = −49 kJ/mol Melting at 100° C. (d)ΔrH_((addition of I) ₂ ₎ = −136 kJ/mol Decomposition at 140° C.step >99.9% ΔrH = −389 kJ/mol Melting at 78° C. (e) No decompositionobserved

Industrial transposition of a synthesis requires easy purification stepsto be preferred, especially the last purification step of the synthesis.

A preferred form of this invention relates to the process of synthesisof compound I as described previously, wherein purification of compoundI is performed by recrystallisation in water.

Industrial transposition of a synthesis requires most stable forms ofcompounds to be preferred, especially the most stable form of the finalproduct.

Studies performed on compound I showed that hydrate forms, particularlytrihydrate form, are more stable than neat form of the compound. Thetrihydrate form compound I, (3H₂O) is the most stable form under ambientconditions. Any mixture of hydrates of compound I will evolve within afew days towards the trihydrate form in ambient conditions. Ambientconditions as used herein refer to a temperature between 15° C. and 25°C., at atmospheric pressure, and a relative humidity rate above 50%.

A preferred form of this invention relates to the process of synthesisof compound I as described previously, wherein compound I is obtained asa hydrate form, preferably as the trihydrate form.

Another object of the present invention is thus the crystallinetrihydrate form of compound I. In particular, the crystallographicstructure of compound I trihydrate is detailed in example 2.

Synthesis of the starting material of the general method describedabove, (S) ethyl 2-(benzyloxycarbonylamino)4-(neopentyloxysulfonyl)butanoate A, has been already described inpatent application WO2004/007441, as an intermediate in the synthesis of4,4′-thiobis(3-aminobutane 1-neopentylsulfonate)bis-trifluoroacetate.Isolation thereof can be readily performed by one of ordinary skill inthe art if necessary.

Another object of the invention is the general method described above,wherein synthesis of compound A from L-Homocystine comprises thefollowing steps:

-   -   (a-1) reacting L-Homocystine with benzyl chloroformate to give        (2S,2S′)        4,4′-disulfanediylbis(2-(benzyloxycarbonylamino)butanoic acid)        F;    -   (b-1) carrying out an esterification reaction between F and        ethanol to give (2S,2S′) diethyl        4,4′-disulfanediylbis(2-(benzyloxycarbonylamino)butanoate) G;    -   (c-1) oxidatively cleaving the disulfide bond of G to give (S)        ethyl 2-(benzyloxycarbonylamino) 4-(chlorosulfonyl)butanoate H;        and    -   (d-1) reacting the sulfonyl chloride H with neopentyl alcohol to        give (S) ethyl 2-(benzyloxycarbonylamino)        4-(neopentyloxysulfonyl)butanoate A.

The synthesis of A from L-Homocystine is illustrated on Scheme 2.

With the same preoccupations as the ones evoked for the synthesis ofcompound I from A, the process to synthesize A may be optimized in orderto make it as much as possible in accordance with industrialrequirements.

Preferably, step (a-1) may be performed in presence of sodium hydroxide.The solvent may be THF. The reaction may be performed at a temperaturefrom about 5° C. to about 25° C., preferably the temperature may remainbetween about 5° C. and about 10° C. during addition of reagents.

Ease of analysis is an important criterion for a synthesis to besuitable for industry. Presence of Cbz amine protective groups oncompound F may make it more appropriate for analysis, particularly forHPLC analysis.

Preferably, step (b-1) may be performed by reacting F with thionylchloride. The solvent may be pure ethanol. The reaction may be performedat a temperature from about 45° C. to about 55° C.

Preferably, step (c-1) may be performed by reacting G with chlorine. Thesolvent may be ethanol. The reaction may be performed at a temperaturefrom about 5° C. to about 10° C.

Preferably, step (d-1) may be performed in presence of triethylamine.The solvent may be toluene. The reaction may be performed at atemperature from about 15° C. to about 25° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Compared XRPD (X Ray Powder Diffraction) patterns of compound Itrihydrate: calculated from single crystal structure (lower spectrum)and experimental (upper spectrum).

FIG. 2: ORTEP (Oak Ridge Thermal Ellipsoid Plot) representation ofcompound I trihydrate.

FIG. 3: Projection along an axis of compound I trihydrate. H-bonds arerepresented by dotted lines.

EXAMPLES Example 1 Synthesis of Compound I from (S) ethyl2-(benzyloxycarbonylamino) 4-(neopentyloxysulfonyl)butanoate A Step (a):(S) neopentyl 3-benzyloxycarbonylamino) 4-hydroxybutane 1-sulfonate B

(S) ethyl 2-(benzyloxycarbonylamino) 4-(neopentyloxysulfonyl)butanoate A(41.55 g, 100.0 mmol, 1.0 eq.) is added dropwise onto a 2M solution ofLiBH₄ in THF (50 mL, 44.8 g, 100.0 mmol, 1.0 eq.). The addition isperformed at room temperature over a 3 hrs period. At the end of theaddition, the mixture is stirred at room temperature until conversion iscomplete (A<1%). Addition of toluene, followed by hydrolysis with HCl,washings of the organic layer with NaHCO₃ and water, and concentrationunder vacuum lead to the desired product as a pale yellow oil inquantitative yield (ee=98%), which slowly crystallises at roomtemperature in 4 or 5 days.

As B was found to have a very low melting point by DSC analysis, it wasnot possible to isolate it as a solid by simple crystallisation. It wasdecided to let it in solution and use it without further purification inthe following step.

Step (b): (S) neopentyl 3-(benzyloxycarbonylamino)4-(methylsulfonyloxy)butane 1-sulfonate C

A solution of B (57.64 g, 154.34 mmol, 1.0 eq.) in toluene (115 mL, 2.0vol.) is diluted with MTBE (173 mL, 3.0 vol.) at room temperature. Mesylchloride (17.9 mL, 26.5 g, 231.50 mmol, 1.5 eq.) is then added at roomtemperature and the homogeneous mixture is cooled to 10° C. The additionof triethylamine (43.0 mL, 31.2 g, 308.67 mmol, 2.0 eq.) is performed atT<20° C. At the end of the addition, the mixture is stirred at 10° C.until conversion is complete (B<1%). After hydrolysis with diluted HCl,the organic layer is washed with NaHCO₃, water and brine, followed by apartial concentration under reduced pressure. The corresponding mesylateis then crystallised by addition of heptanes (5.0 vol.) at 40° C. Aftercooling, filtration and drying, the expected product is isolated as awhitish solid in 92.5% yield and with a very high chemical purity (98%).

Step (c): (S) 2-(benzyloxycarbonylamino) 4-(neopentyloxysulfonyl)butylthioacetate D

A solution of mesylate C (81.3 g, 180.05 mmol, 1.0 eq.) in acetone (203mL, 2.5 vol.) is added dropwise to a suspension of potassium thioacetate(41.1 g, 360.1 mmol, 2.0 eq.) in acetone (203 mL, 2.5 vol.) at roomtemperature and over a period of 2 hrs. The reaction mixture is stirredat room temperature until conversion is complete (C<1%). Afterfiltration of the salts and addition of toluene (4.0 vol.), acetone isremoved by distillation under reduced pressure at 25° C. The solution isthen treated with active charcoal and concentrated to 2.0 volumes. Slowaddition of heptane (5.0 vol.) at room temperature, followed by coolingat 0° C., filtration and drying at 45° C., provides the expected productas a whitish solid in 78.2% yield and with a very high chemical purity(98%).

Step (d): (3S,3S′) neopentyl4,4′-disulfanediylbis(3-(benzyloxycarbonylamino)butane 1-sulfonate) E

A solution of D (59.16 g, 137.1 mmol, 1.0 eq.) suspended in ethanol (203mL, 2.5 vol.) is cooled to 0° C. 20% sodium hydroxide (25.1 mL, 150.8mmol, 1.1 eq.) diluted with water (16.9 mL, 0.285 vol.) is then addeddropwise to the suspension by keeping the temperature below 10° C. Thereaction mixture is warmed to room temperature and stirred untilconversion is complete (D<1%). The intermediate thiol reacts at roomtemperature with a solution of iodine (20.9 g, 82.3 mmol, 0.6 eq.) inethanol (118 mL, 2.0 vol.). The reaction is complete at the end of theaddition of the oxidizing agent. After addition of a Na₂S₂O₅ (13.0 g,68.5 mmol, 0.5 eq.) aqueous solution (118 mL, 2.0 vol.) to reduce theexcess of residual iodine, ethanol is removed by distillation underreduced pressure at 40° C. Addition of water (3.0 vol.) at roomtemperature, followed by cooling at 0° C., filtration and drying at45-50° C., provides the expected dimer as a white solid in 98.3% yieldand with a very high chemical purity (97.0%). The amount of iodide ions,coming from the reduction of iodine, is checked in the sample bypotentiometric assay.

-   E⁰(Ag⁺/Ag(s))=0.80V-   Ks_(AgI)=1.5.10⁻¹⁶-   [AgNO₃]=0.1N-   Electrode: E=E⁰(Ag⁺/Ag(s))+0.06 log [Ag⁺]-   [Ag⁺]=K_(SI)/[I⁻]-   E=E° (Ag⁺/Ag(s))+0.06 log (K_(SI)/[I⁻])-   Assay: [I⁻] decreases and E increases-   LOD=1 mg

Four further washings with water are performed until no more iodide ionsare detected. The results are presented in table 2.

TABLE 2 Washings 1 2 3 4 I⁻assay (%) 4.5 1.26 0.12 0.02

Step (e): (3S,3S′) 4,4′-disulfanediylbis(3-aminobutane 1-sulfonic acid)compound I

A solution of E (44.0 g, 56.6 mmol, 1.0 eq.) in TFA (220 mL, 5.0 vol.)and anisole (44 mL, 1.0 vol.) is heated to reflux (75° C.) and thereaction mixture is stirred in these conditions until conversion iscomplete (E<1%). TFA is removed by distillation under reduced pressureat 50° C. Slow addition of MTBE (5.0 vol.) at room temperature makes theexpected product precipitate. After trituration, filtration and washingwith MTBE (1.0 vol.), the crude solid is suspended in methanol (220 mL,5.0 vol.). New trituration, filtration and washing with MTBE (1.0 vol.),followed by drying under reduced pressure, provides compound I as awhite solid in 92.5% yield.

NMR: ¹H (solvent D₂O, 400 MHz, ppm): 4.70 (s, 6H, H₅); 3.77 (m, 2H, H₂);3.14 (dd, 2H, H₁); 2.98 (dd, 4H, H₄); 2.86 (dd, 2H, H₁); 2.13 (m, 4H,H₃). ¹³C (solvent D₂O, 100 MHz, ppm): 49.4 (2C, C₂); 46.6 (2C, C₄); 38.3(2C, C₁); 26.9 (2C, C₃).

Example 2 Crystallographic Data of Compound I Trihydrate

Compound I obtained in example 1 was stored for 22 days in ambientconditions in order to exclusively contain trihydrate form.

Data Collection:

The crystal structure of compound I trihydrate [C₈S₄N₂O₆H₂0, 3(H₂O)],has been determined from single crystal X-Ray diffraction (XRD). Thechosen crystal was stuck on a glass fibre and mounted on the fullthree-circle goniometer of a Bruker SMART APEX diffractometer with a CCDarea detector. Three sets of exposures (a total of 1800 frames) wererecorded, corresponding to three ω scans (steps of 0.3°), for threedifferent values of φ.

FIG. 1 shows experimental and calculated XRD patterns of compound Itrihydrate.

Table 3 presents a selection of calculated reflections from PowderCellfor the compound I trihydrate structure and the correspondingexperimental peaks positions and intensities. Intensity values ofexperimental peaks must not be conferred too much importance sincepreferential orientation effects relying on the crystallization processare very important.

TABLE 3 2θ (°) d (Å) I (%) 2θ (°) d (Å) I (%) h k l CalculatedExperimental 0 0 1 5.08 17.39 14 5.09 17.34 76 0 1 2 14.27 6.20 23 14.306.19 23 0 0 3 15.28 5.79 7 15.30 5.79 100 −1 0 2 17.60 5.03 13 17.625.03 15 0 1 3 18.28 4.85 55 18.32 4.84 96 1 0 2 18.59 4.77 40 18.61 4.7630 1 1 1 18.95 4.68 28 18.98 4.67 16 −1 1 2 20.27 4.38 47 20.34 4.36 260 2 1 20.70 4.29 37 20.75 4.28 26 1 1 2 21.15 4.20 14 21.16 4.19 11 1 03 22.05 4.03 27 22.09 4.02 37 0 1 4 22.77 3.90 12 22.79 3.90 37 −1 1 323.11 3.85 3 23.12 3.84 6 −1 0 4 24.66 3.61 47 24.65 3.61 74 1 2 0 25.103.54 13 25.15 3.54 7 0 0 5 25.60 3.48 4 25.60 3.48 62 1 2 1 25.80 3.45100 25.83 3.45 39 −1 1 4 26.67 3.34 5 26.66 3.34 7 0 1 5 27.54 3.24 227.52 3.24 13 1 1 4 28.01 3.18 1 28.05 3.18 4 −1 0 5 28.98 3.08 1 28.943.08 4

The cell parameters and the orientation matrix of the crystal werepreliminary determined by using SMART Software. Data integration andglobal cell refinement were performed with SAINT Software. Intensitieswere corrected for Lorentz, polarisation, decay and absorption effects(SAINT and SADABS Softwares) and reduced to F_(o) ². The program packageWinGX3 was used for space group determination, structure solution andrefinement.

Data Refinement:

The standard space group P2₁ (n°4) was determined from systematicextinctions and relative F_(o) ² of equivalent reflections. Thestructure was solved by direct methods (SIR 92). Anisotropicdisplacement parameters were refined for all non-hydrogen atoms.Hydrogen atoms were located from subsequent difference Fourier synthesesand placed with geometrical constraints (SHELXL). The final cycle offull-matrix least-square refinement on F2 was based on 3714 observedreflections and 234 variable parameters and converged with unweightedand weighted agreement factors of:

-   R1=0.0347, wR2=0.0845 for [F²>2σ(F²)] and R1=0.0371, wR2=0.0934 for    all data.

The crystals were obtained by slow evaporation of a saturated solutionof compound I in water at ambient temperature.

Crystallographic Data:

TABLE 4 Chemical Formula C₈S₄N₂O₆H₂₀, 3(H₂O) Molecular Weight/g · mol⁻¹422,55 Crystal System Monoclinic Space Group P2₁ (n°4) Z 2 Z’(asymmetric units per unit cell) 1 a/Å 5.936 (2) b/Å 8.849 (3) c/Å17.416 (7) β (°) 93.349 (6) V/Å³ 913.4 (6) dcalc/g · cm⁻³ 1.536Temperature/K 293 (2) Absolute structure parameter 0.0 (5) Crystalcolour colourless Approximate crystal size/mm 0.5 × 0.3 × 0.05 F(000)/e−448 Absorption coefficient μ (MoKα1)/mm⁻¹ 0.562

Table 5 presents the atomic coordinates (×10⁴) and equivalent isotropicdisplacement parameters (Å²×10³). U(eq) is defined as one third of thetrace of the orthogonalized Uij tensor.

TABLE 5 x y z U(eq) S(1) −6907 (8) 11820 (6) 2086 (3) 36 (1) S(2) −120(8) 6540 (6) 3958 (3) 29 (1) S(1A) −5005 (10) 13527 (7) 2563 (3) 36 (1)S(2A) 1135 (8) 14087 (6) −658 (3) 28 (1) O(1) 1610 (20) 7004 (18) 3451(9) 39 (4) O(2) −1390 (30) 5256 (19) 3657 (11) 46 (4) O(3) 800 (30) 6300(20) 4737 (9) 52 (5) O(1A) 2310 (30) 12720 (20) −396 (12) 53 (5) O(2A)2380 (30) 15414 (19) −407 (9) 39 (4) O(3A) 510 (30) 14090 (30) −1476 (9)53 (5) N(1) −6330 (30) 9880 (20) 3645 (10) 31 (4) N(1A) −4670 (30) 15277(19) 942 (9) 28 (4) C(1) −5210 (40) 10160 (30) 2306 (12) 35 (5) C(2)−4410 (30) 9920 (20) 3132 (11) 28 (4) C(3) −3070 (40) 8440 (30) 3193(12) 35 (5) C(4) −2020 (40) 8090 (20) 3988 (12) 31 (4) C(1A) −2670 (40)13690 (30) 1940 (12) 35 (5) C(2A) −3260 (30) 13880 (20) 1091 (11) 27 (4)C(3A) −1110 (30) 13990 (30) 661 (11) 29 (4) C(4A) −1460 (30) 14090 (30)−199 (11) 31 (4) OWA 2000 (30) 6520 (20) 1873 (11) 51 (5) OWB −9670 (40)12120 (20) 3802 (12) 56 (5) OWC −5190 (40) 9820 (30) 5188 (12) 70 (7)Structural Description:

The asymmetric unit is composed of a single compound I moleculeassociated with 3 molecules of water. FIG. 2 presents the asymmetricunit of the molecule of compound I and the 3 molecules of water.

Along b axis, successive molecules of compound I interact via two kindsof hydrogen bonds established between the oxygen atom O1A and thehydrogen atom H(N1A) (d˜1.94 Å), and between the oxygen atom O1 and thehydrogen atom H(N1) (d˜1.99 Å). Along a axis, two consecutive moleculesof compound I interact via a hydrogen bond between the oxygen atom O2Aand the hydrogen atom H(N1A) (d˜1.98 Å). These interactions oriented ina and b directions lead to layers parallel to (110). Furthermore, awater molecule (OWA) is inserted between these molecules and establishesthree different hydrogen bonds: the first one links the oxygen atom OWAto the hydrogen atom H(N1A) (d˜2.02 Å), the second one links the oxygenatom O3A to the hydrogen atom H(OWA) (d˜1.94 Å) and the last one linksthe oxygen atom O1 to H(OWA) (d˜1.97 Å). The slices (110) present athickness of d₀₀₁ (˜17.5 Å). The different interactions inside theseslices along a axis are represented more specifically on FIG. 3. Twoconsecutive layers interact along c axis through hydrogen bondsestablished with the two others water molecules OWB and OWC located in(002) planes. The oxygen atom of the OWB water molecule establisheshydrogen bonds with the hydrogen atom H (from N1) (d˜2.00 Å), from afirst slice, and is connected to the following slice via a hydrogen bondwith the oxygen atom O3 and the hydrogen atom H (from OWB) (d˜1.92 Å).The oxygen atom of the OWC water molecule establishes a hydrogen bondwith the hydrogen atom H (from N1) (d˜1.86 Å) for a first slice, andthen is linked to the adjacent slice by the way of two hydrogen bondsthrough interactions of two oxygen atoms from the sulfonate moiety S2.The oxygen atom O2 and the oxygen atom O3 interact with the two hydrogenatoms of the water molecule OWC with bond lengths of respectively d˜2.30Å and d˜2.03 Å.

Example 3 Synthesis of (S) ethyl 2-(benzyloxycarbonylamino)4-(neopentyloxysulfonyl)butanoate A from L-homocystine Step (a-1):(2S,2S′) 4,4′-disulfanediylbis(2-(benzyloxycarbonylamino)butanoic acid)F

L-Homocystine (200.0 g, 745.5 mmol, 1.0 eq.) is suspended in THF (1000mL, 5.0 vol.) and cooled to 5-10° C. Addition of 20% sodium hydroxide(521.7 mL, 626.2 g, 125.25 g at 100%, 3.13 mmol, 4.2 eq.) is followed byaddition of benzyl chloroformate (220.3 mL, 267 g, 1565.5 mmol, 2.1eq.). Conversion is complete after a night at room temperature(L-Homocystine<1%). Extractions and washings of the organic layer withwater leads to a yellow oil which is isolated in quantitative yield, andwhich slowly crystallizes as a yellowish solid at room temperature.

Step (b-1): (2S,2S′) diethyl4,4′-disulfanediylbis(2-benzyloxycarbonylamino)butanoate) G

F (140.0 g, 260.89 mmol, 1.0 eq.) is suspended in pure ethanol (700 mL,5.0 vol.) and heated to 50° C. The addition of SOCl₂ (41.6 mL, 68.3 g,573.96 mmol, 2.2 eq.) is run at 50° C. to avoid accumulation. Conversionis complete (F<1%) after 1 hr at 50° C. Concentration of the crudemixture followed by dissolution in ethyl acetate and washings of theorganic layer leads to a clear solution which is partially concentrated.Slow addition of 5.0 volumes of heptane leads to crystallisation of thedesired product as a white solid. After filtration and drying, thebis-ester is isolated in 92% yield.

Step (c-1): (S) ethyl 2-benzyloxycarbonylamino)4-(chlorosulfonyl)butanoate H

G (100.0 g, 168.7 mmol, 1.0 eq.) is suspended in ethanol (500 mL, 5.0vol.) and cooled to 5° C. The addition of Cl₂ (83.7 g, 1.18 mol, 7.5eq.) is performed at T<10° C. Conversion is complete (G<1%) when thereaction mixture is perfectly homogeneous. The sulfonyl chloridesolution is poured onto a mixture of an aqueous carbonate solution andtoluene, keeping the temperature below 20° C. Washings of the organiclayer, followed by concentration under reduced pressure, lead to thedesired product as a colorless oil in 96.8% yield.

The expected product can be isolated as a white solid when 5.0 volumesof heptane are slowly added to a concentrated toluene solution (2.0vol.) of the product. Nevertheless, the purity is not really improvedand the yield dramatically decreases (75-80%). Consequently, thesulfonyl chloride H is isolated in 1.0 volume of toluene and usedwithout further purification in the following step.

Step (d-1): (S) ethyl 2-(benzyloxycarbonylamino)4-neopentyloxysulfonyl)butanoate A

Neopentyl alcohol (29.1 g, 329.84 mmol, 1.2 eq.) is dissolved in toluene(400 mL, 4.0 vol.) and a solution of H (100.0 g, 274.87 mmol, 1.0 eq.)in toluene (100 mL, 1.0 vol.) is added at room temperature. Thehomogeneous mixture is then cooled to 0° C. The addition oftriethylamine (46.0 mL, 33.4 g, 329.84 mmol, 1.2 eq.) is performed at 0°C. At the end of the addition, the mixture is warmed to room temperatureuntil conversion is complete (H<1%). After hydrolysis with dilute HCl,the organic layer is washed with NaHCO₃, water and brine, andconcentrated under reduced pressure to give the desired product as apale yellow oil in 94.4% yield.

The invention claimed is:
 1. A process for preparing (3S,3S′)4,4′-disulfanediylbis(3-aminobutane 1-sulfonic acid) from (S) ethyl2-(benzyloxycarbonylamino) 4-(neopentyloxysulfonyl)butanoate Acomprising the steps of: (a) reducing the ethyl ester of A to give (S)neopentyl 3-(benzyloxycarbonylamino) 4-hydroxybutane 1-sulfonate B; (b)reacting the alcohol B with methanesulfonic anhydride or methanesulfonylchloride in presence of a base to give (S) neopentyl3-(benzyloxycarbonylamino) 4-(methylsulfonyloxy)butane 1-sulfonate C;(c) reacting the mesylated alcohol C with potassium thioacetate to give(S) 2-(benzyloxycarbonylamino) 4-(neopentyloxysulfonyl)butyl thioacetateD; (d) dimerizing D to give (3S,3S′) neopentyl4,4′-disulfanediylbis(3-(benzyloxycarbonylamino)butane 1-sulfonate) E;and (e) deprotecting sulfonic ester and amine groups of E to give(3S,3S′) 4,4′-disulfanediylbis(3-aminobutane 1-sulfonic acid).
 2. Theprocess according to claim 1, wherein step (a) is performed by reactingA with a reducing agent—solvent couple selected from NaBH₄/LiCl—mixtureof THF and ethanol and LiBH₄—THF, at a temperature from about 0° C. toabout 25° C.
 3. The process according to claim 1, wherein step (a) isperformed by reacting A with LiBH₄—THF, at a temperature from about 20°C. to about 25° C.
 4. The process according to claim 1, wherein step (b)is performed in presence of triethylamine as base, in a solvent selectedfrom chloroform and a mixture of MTBE and toluene, at a temperature fromabout −10° C. to about 10° C.
 5. The process according to claim 1,wherein step (b) is performed by reacting B with methanesulfonylchloride, in presence of triethylamine as base, in a mixture of MTBE andtoluene in 3:2 volume ratio, at a temperature from about 5° C. to about10° C.
 6. The process according to claim 1, wherein step (c) isperformed in a solvent selected from ethanol and acetone.
 7. The processaccording to claim 1, wherein step (c) is performed in acetone, at atemperature from about 15° C. to about 25° C.
 8. The process accordingto claim 1, wherein step (d) is performed by contacting D with sodiumhydroxide, in ethanol, and reacting the obtained mixture with iodine, inethanol, at a temperature from about 15° C. to about 25° C.
 9. Theprocess according to claim 1, wherein step (e) is performed by stirringE in a mixture of TFA and anisole.
 10. The process according to claim 1,wherein step (e) is performed by stirring E in a refluxing mixture ofTFA and anisole, in 5:1 volume ratio.
 11. The process according to claim1, wherein purification of the final product is performed byrecrystallisation in water.
 12. The process according to claim 1,wherein the final product is obtained as one of its hydrate forms. 13.The process according to claim 1, wherein A is prepared fromL-Homocystine by a process comprising the steps of: (a-1) reactingL-Homocystine with benzyl chloroformate to give (2S,2S′)4,4′-disulfanediylbis(2-(benzyloxycarbonylamino)butanoic acid) F; (b-1)carrying out an esterification reaction between F and ethanol to give(2S,2S′) diethyl4,4′-disulfanediylbis(2-(benzyloxycarbonylamino)butanoate) G; (c-1)oxidatively cleaving the disulfide bond of G to give (S) ethyl2-(benzyloxycarbonylamino) 4-(chlorosulfonyl)butanoate H and (d-1)reacting H with neopentyl alcohol to give (S) ethyl2-(benzyloxycarbonylamino) 4-(neopentyloxysulfonyl)butanoate A.
 14. Theprocess according to claim 13, wherein step (a-1) is performed in THF inpresence of sodium hydroxide, at a temperature from about 15° C. toabout 25° C.
 15. The process according to claim 13, wherein step (b-1)is performed by reacting F with thionyl chloride, in pure ethanol, at atemperature from about 45° C. to about 55° C.
 16. The process accordingto claim 13, wherein step (c-1) is performed by reacting G withchlorine, in ethanol, at a temperature from about 5 to about 10° C. 17.The process according to claim 13, wherein step (d-1) is performed inpresence of triethylamine, in toluene, at a temperature from about 15°C. to about 25° C.
 18. The process according to claim 1, wherein thefinal product is obtained as its trihydrate form.